After 4 rounds of biopanning against a tumor antigen (biotinylated and bound to streptavadin beads) elisas done with scfv that leaked into the culture supernatant show no signals. Western blot shows that colonies of infected Tg1 cells are not expressing any SCFV. Expression of scfv into supernatant depends on leaky amber stop codon between scfv and phage G3 protein. Would one still expect scfv production (of off target scfv's such as strepavadin binders) if biopanning had failed to enrich for positive binders? Would a non amber suppressor such as ss320 be suitable for protein expression/ potentially give higher scfv yeild?
Commonly IPTG induction is used to produce scFv, unlike phage display that normally relies on leaky expression. Are you doing that?
You can still try to do 96 well phage ELISA to determine whether the panning totally failed or not. If you have only negative phages its likely that panning have failed and output clones can even be the ones not able to produce any scFv.
On the other hand, if phages are positive, but soluble scFv production fails this can be related to a deficit in secretion of soluble scFvs not fused to PIII. This can be related to aggregation problems or to other reasons.
In any case, you can try to express the soluble molecules in a non-suppressor strain to increase the expression levels, but also you can reclone the genes in an optimized vector for scFv production. Changing vector/host (for instance using chaperone- containing vectors and BL-21 strain) can improve yields of soluble scFv. You can even try mammalian cell expression to allow a proper folding and secretion.
Its always good to sequence at least some selected clones. That can reveal the presence un unwanted amber stop codons having little effect on very sensitive phage ELISA, but a huge effect on soluble scFv production, and other artifacts.
Thank you for your answer. Yes I do induce with IPTG 1mM and using this concentration can induce scfv expression in colonies from other members of my lab. I am trying to get phage elisas to work, do you have an antibody that works well for a phage elisa? I saw most people have used a anti m-13 antibody from GE but that was discontinued.
I still have the GE anti-M13/HRP. I know it will be necessary to replace it with an avilable product, but I dont have yet any advice about that.
Colony PCR is showing me that only some of the colonies I am picking for ELISA are infected with phage. I upped the ampicillin concentration in my plates to 300 ug/ml but still 2/3 colonies with no phage. Where are these other colones coming from? I don't think they are satellites because I am picking only the largest colonies farthest away from other colones. Any ideas?
How is your colony PCR designed? How can yo be sure that you have clones without any phagemid and not false-negative colony PCR results?
The primers I have come from the distributor of the plasmid. The positive bands look very different (from the false negative bands) when they show up, much larger. I run every pcr with a sample of dna I know contains the plasmid and compare to that. Additionally I have sequenced the dna and positive bands have the correct sequence.
So your negative bands come from phagemids without an insert, is that right? If so, its not surprising that colonies with such plasmids grow in the presence of amp, they are resistant, but have no dont have any foreign gene.
Firstly, thanks for the help Gertrudis. No, the negative bands are distinct from phagemids without an insert. My primers flank the cloning site and I can see some phagemids without an insert showing up as distinct bands 700 base pairs below the phagemid bands with the insert. Here is one of my gels attached for reference.
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